Construction of VSMC-Cx43^-/- conditional knockout mouse model and genotype identification

Objective To construct a conditional gene knockout mouse (VSMC-Cx43^-/-) model of vascular smooth muscle connexin Cx43 by using CRISPR-Cas9 technology and LoxP-Cre system.

Methods Using the principle of homologous recombination, the Gja1 gene was modified with flox by homologous recombination of spermatovum. Cas9 mRNA and gRNA were obtained by in vitro transcription; the homologous recombination vector was constructed by the In-Fusion cloning. The vector contained a 3.0 kb 5'homology arm, a 1.9 kb flox region and a 3.0 kb 3'homology arm. Cas9 mRNA, gRNA and donor vector were microinjected into the spermatovum of C57BL/6J mice to obtain F0 generation mice. The positive F0 generation mice identified by PCR amplification and sequencing were mated with C57BL/6J mice to obtain 3 positive F1 generation mice (Cx43^flox/+). The obtained flox heterozygous Cx43^flox/+ mice were selfed to obtain flox homozygous Cx43^flox/flox mice. Then use Cx43^flox/flox mice and Myh11-CreERT2 mice to mate, and finally obtain the experimental group mice that are flox homozygous and Cre-positive (the mouse is abbreviated as: VSMC-Cx43^-/-) and flox homozygous and Cre negative/control mice (Cx43^flox/flox). PCR was used for genotype identification, and Western blot was used to detect the expression of Cx43 in the middle cerebral artery, coronary artery, pulmonary artery and renal artery tissues to determine whether the gene knockout mice were successfully constructed.

Results Genotype identification, Immunofluorescence, and WB detection result showed that the VSMC-Cx43^-/- gene knockout mouse model was successfully constructed. The general characteristics of the mice remained unchanged, and the expression of Cx43 protein in the middle cerebral artery, coronary artery, pulmonary artery and renal artery tissues of the mouse decreased. Therefore, it can be used as a long-term stable model to study the function of vascular smooth muscle Cx43.

Conclusion The VSMC-Cx43^-/- gene knockout mouse model was successfully constructed using CRISPR-Cas9 technology and LoxP-Cre system, which provides a powerful tool for in-depth study of the role of Cx43 in vascular diseases.